A rat hybridoma model of Heymann nephritis: production of a monoclonal anti GP330 from a nephritic rat.
Autologous (Heymann) nephritis was induced by immunizing Sprague-Dawley rats with a crude membrane extract (Fx1A) prepared from renal cortical tubules. Urine protein excretion was monitored to determine the onset of nephritis and then the spleens of nephritic rats were fused with a non-secretor rat myeloma cell line. Supernatants from hybridoma cultures were first screened for production of anti-brush border membrane (BBM) antibody by immunodot blotting of highly purified rat BBM on nitrocellulose. Positive hybrids were then tested by indirect immunofluorescence for the presence of IgG, which binds to the brush border of rat renal proximal tubules. Those hybrids which were positive by both screening assays were subcloned twice. Two monoclonal antibodies (C5, D11) were studied in some detail. Both C5 and D11 immunoprecipitated a single polypeptide from BBM, labelled with 125I by the lactoperoxidase method. Radioautography of gradient 4-11% slab sodium dodecyl sulphate polyacrylamide gels revealed that the polypeptide against which C5 and D11 were directed co-migrated with the polypeptide immunoprecipitated by (i) IgG eluted from the renal cortex of nephritic rats, and by (ii) a mouse anti-rat monoclonal against gp 330 [a BBM constituent with proven pathogenicity [9]]. Supernatants which tested positive by immunodot blotting but negative by indirect immunofluorescence showed no detectable immunoprecipitate after reaction with BBM. Immunocytochemical staining by immunoperoxidase and immunogold methods localized C5 and D11 to the BBM of the renal proximal tubule and to the urine face of the glomerular epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)
Behar
M
M
Department of Medicine, University of Toronto.
Katz
A
A
Silverman
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000941:Antigens / Q000276:immunology; D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D005921:Glomerulonephritis / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D007687:Kidney Tubules, Proximal / Q000276:immunology; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D000941:Antigens; D006023:Glycoproteins